热力学因素对绵羊重组朊蛋白体外构象转化的作用  

Effect of the thermodynamic factor on conformational conversion of the ovine prion protein in vitro

作　　者：王伊琴[1,2] 秦贞奎[3] 包勇敢 乔俊文[1] 赵德明[1] 

机构地区：[1]中国农业大学动物医学院/国家动物传染性海绵状脑病实验室,北京100193 [2]内蒙古二连浩特出入境检验检疫局,内蒙古二连浩特011100 [3]中国检验检疫科学研究院,北京100123

出　　处：《中国农业大学学报》2009年第4期21-27,共7页Journal of China Agricultural University

基　　金：国家自然科学基金资助项目(30871854);国家科技支撑计划资助项目(2006BAD06A13)

摘　　要：构建了绵羊朊蛋白的原核表达载体,获得了高纯度的融合表达蛋白。并在热力学因素的作用下,研究了重组绵羊朊蛋白的构象转化。以基因型为ARQ/ARQ蒙古绵羊的血液DNA为模板,利用DNA重组技术,将绵羊朊蛋白正常成熟蛋白基因OvPrP插入表达载体pET30a,在大肠杆菌BL2l(DE3)中高效表达,获得的表达产物以包涵体形式存在,并对其进行纯化和复性。超滤浓缩后浓度约为0.5 mg/mL的OvPrP97-234,进行热力学处理,利用远紫外线圆二色谱(CD)分析热力学处理前、后蛋白的二级结构的变化,同时,对热力学处理前、后的蛋白进行了蛋白酶K抗性的检测,并对其高级结构进行了预测。结果表明:获得的表达产物经SDS-PAGE分析可见分子量为16kD的蛋白条带,Western-blotting的鉴定证实了所获得的蛋白是特异性的朊蛋白。经Jascow32软件分析,测得天然构象的OvPrP97-234的二级结构含量为:α螺旋为28.8%、β折叠为0%、转角和无规卷曲为71.1%,无蛋白酶K的抗性。经过热力学处理之后,OvPrP97-234的二级结构含量为:α螺旋为19.3%,β折叠为36.9%,转角和无规卷曲为43.8%,有一定的蛋白酶K抗性。绵羊重组朊蛋白的体外构象转化的分析为朊蛋白的体外构象转化机制和致病机理的研究提供科学依据。This study obtained fusion protein with high purity of ovine prion protein, and constructed the recombinant prokaryotic expression vector for ovine prion protein gene . It investigated the conformational conversion of ovine priori protein by the effect of the thermal denaturation in vitro. The ovine prion protein gene（Pmp） was expressed in E. coli. by the DNA recombination. The OvPrPc occurred in the sensitive cells （BL2 1） by the denatured inclusion body. After purification and refoldation of the recombinant OvPrPc, conformational changes of ovine prion protein was triggered by thermal denaturation. The results showed that molecular weight of the target protein was approximately 15 767.3 D and it possessed the antigentic activity of OvPrP97-234. Circular dichroism spectra showed that the content of structure was α helix （28. 8%） ,β sheet （0）, turn and freedom curl （71. 1%）, digested by Proteinase K. After thermal denaturation ,conformational changes of the ovine prion protein was shown in CD spectra. The content of structure was α helix （19.3%）, β sheet （36.9%）, tum and freedom curl （43.8%）. Proteinase K digestion assay showed the proteinase-resistance. The finding indicated the consequent molecular mechanisms of PrP conversion triggered by thermal denaturation, and the information gained from the present study may provide new insight into the mechanism of prion diseases.

关 键 词：绵羊重组朊蛋白 原核表达 构象转化 圆二色谱 热力学因素 

分 类 号：S852.659.7[农业科学—基础兽医学] Q786[农业科学—兽医学]